Identifying Decomposition Products in Extracts of Cellular

Most methods of analyzing intracellular metabolites require extraction of metabolites from the cells. A concern in these methods is underestimation of metabolite levels due to incomplete extraction. In comparing extraction methods, it would accordingly seem that the best method for extracting a particular metabolite is the one giving the largest yield. In extracting Escherichia coli with different methanol:water mixtures, we observed that ≥ 50 % water gave increased yield of nucleosides and bases compared to ≤ 20 % water, as determined by liquid chromatography-tandem mass spectrometry analysis of the resulting extracts. Spiking of the extracts with isotope-labeled nucleotides revealed, however, that the high yield of nucleosides and bases occurred due to decomposition of nucleotides in the water-rich condition, not good extraction. Spiking combined with isotope labeling provides a general approach to detecting decomposition products in extracts of cellular metabolites. For extraction of E. coli with methanol:water, cold temperature and a high methanol fraction minimizes artifacts due to metabolite decomposition